The present invention relates to eggs comprising a synergistic composition of antioxidants and low amounts of poly-unsaturated fatty acids (PUFA) (hereinafter called "low PUFA") for reducing the oxidation stress of LDL-cholesterol (hereinafter called "LDL"), to a method for their production and to their consumption in human diet designed to reduce the responsive increase of LDL oxidation associated with consumption of prior art eggs which are higher in polyunsaturated fatty acids.
Researchers all over the world proved the link between heart disease and high blood cholesterol, especially high LDL and high LDL/HDL ratio which is accepted as "risk factor".
Early experiments in animals and humans showed that diets comprising large amounts of saturated fatty acids (SFA) and cholesterol increase the risk of high blood cholesterol and cardiovascular (CV) conditions. This lead to the "lipid hypothesis", suggesting that atheroscerosis is caused by hypercholesterolemia-induced deposition of lipids in the vessel wall and to the public recommendation to restrict the cholesterol consumption to 300 mg/day and to reduce the SFA consumption, especially from animal source.
The arithmetic manifestation of this approach is defined by "the cholesterol saturated fat index (CSI) for coronary prevention; background, use and a comprehensive table of foods", Connor et al., J. Am Diet Assoc., 1989, June 89 (69), pp. 807-16. According to this approach low CSI, i.e., food low in SFA and/or cholesterol content, is considered to have a hypocholesterolemic and therefore low atherogenic potential.
As egg yolk contains both 213-240 mg of cholesterol and "animal fat" eggs were among the first food to be excluded from the diet of western countries consumers.
Modifying the egg quality by changing the chicken's diet was challenged by many researchers, trying to invent an egg compatible with low CSI, namely having low cholesterol and low SFA content, in order to prevent the increase of blood cholesterol and SFA in human consumers.
There have been performed many researches in order to produce an egg which would solve the above problem. The results of these researches are described, e.g., in the following Patents: U.S. Pat. No. 4,187,294, U.S. Pat. No. 4,394,376. Ca U.S. Pat. No. 1,115,983, U.S. Pat. No. 4,410,541, U.S. Pat. No. 4,197.293, U.S. Pat. No. 4,197,294, U.S. Pat. No. 4,128,640, U.S. Pat. No. 5,012,761, U.S. Pat. No. 4,738,853, U.S. Pat. No. 4,868,001, U.S. Pat. No. 3,657,423 and U.S. Pat. No. 5,246,717.
These patents substantially describe and claim eggs compatible with a cholesterol-reducing diet and methods of their production. The methods described comprise increasing the iodine and the PUFA content in the chicken's food and therefore of the laid eggs.
In U.S. Pat. No. 5,246,717 it is indicated that for eggs to be compatible with cholesterol reducing diet they should contain not more than 34% of SFA. No differentiation was made between the amounts of the various unsaturated fatty acids, namely between PUFA and mono-unsaturated fatty acids (MUFA) present. Accordingly, the discussion related to chicken feed in U.S. Pat. No. 5,246,717 concerns feed that results in low SFA. No discussion is disclosed in U.S. Pat. No. 5,246,717 concerning feed which results in controlled amounts of PUFA and/or MUFA.
All the above publications relate to the blood cholesterol concentration, which is compatible with the "lipid hypothesis".
However, a paper of Brown et al., J. Am. Diet Assoc. 46, pp. 189-192, 1965, which is mentioned in U.S. Pat. No. 5,246,717 (column 5, lines 62-67) states that: ". . . consumption of modified eggs rich in polyunsaturated fat was ineffective in reducing serum cholesterol . . . ". This means that neither the inventors of the invention described and claimed in U.S. Pat. No. 5,246,717, nor the authors of the publication cited therein were aware of the fact that a large amount of PUFA in the egg may have a bad effect on the blood cholesterol and in particular on the oxidizability of the LDL, as will be shown hereinafter.
It has also recently been found that only those people responding to dietary cholesterol, known as "responders", are sensitive to external/dietary cholesterol. In "non-responders", the external cholesterol consumed operates effectively the feed-back mechanism to keep cholesterol within normal range. This individual variation could explain the contradictory results in studies on the dietary effect on the blood cholesterol concentration.
Another competing hypothesis of atherosclerosis is the "response to injury hypothesis". This explains the first phase, the initiation of the atherogenic process. Much evidence suggest that lipid oxidized products (LOPS) obtained either as a result of a diet or formed in vivo both initiate and promote the process, i.e., cholesterol oxidized products (COPS) and/or oxidized LDL are move atherogenic than the non-oxidized form. Thus, low rancidity, un-oxidized lipids and a large amount of antioxidants reduce the production of lipids and of LDL oxidation. Here PUFA due to its great reactivity to oxidation differs greatly from MUFA which is rather resistant.
As easily can be understood from the above, although there exists a wide agreement that the LDL oxidation in the blood is a major risk for arteriosclerosis, so for no research regarding the correlation between eating eggs and the sensitivity of LDL to oxidation has been performed.
However, a large amount of resources have been spent on fining a correlation between the oxidation of LDL to arteriosclerosis and the reasons causing said oxidation and methods of preventing same. This can be seen, e.g., in the following publications:
1. "Low density lipoprotein rich in oleic acid is protected against oxidative modification: Implications for dietary prevention of atherosclerosis" Sampath Parthasarthy et al. Proc. Nat. Acad. Sci. USA; Vol. 87 pp. 3894-3898, May 1990 Medical Sciences. PA0 2. "Inverse correlation between plasma vitamin E and morality from ischemic heart disease in cross-cultural epidemiology" K. Fred Gey et al., Am. J. Clin. Nutr. 1991; 53:626S-34S. 1991 USA. PA0 3. "Antioxidant Vitamins and Low-density Lipoprotein Oxidation" Abbey, M., Nestel, P. J. Et al; Am. J. Clin. Nutr. 58:525-532, 1993, USA. PA0 4. "Comparative Study on the Effect of Low-Dose Vitamin E and Probucol on the Susceptibility of LDL to Oxidation and the Progression of Atherosclerosis in Watanabe Heritable Hyperlipidemic Rabbits", Kleinveld, et al; Arterioscler. Thromb. 14:1386-1391, 1994. PA0 5. "Increase ion Oxidation Resistance of Atherogenic Serum Lipoproteins Following Antioxidant Supplementation: A Randomized Double Blind Placebo-Controlled Clinical Trial" Nyyssonen, K., et al. EUR. J. Clin. Nutr. 13, 1994. PA0 6. "The Effect of Alpha-tocopherol Supplementation on LDL Oxidation" Jialal, I., et al. Arterioscler. Thromb. Vasc. Biol. 15:190-198, 1995. PA0 7. "Dietary Supplementation with Vitamins C and E inhibits in Vitro Oxidation of Lipoproteins" Rifici, V. A. et al., J. AM. Coll. Nutr. 12:631-637, 1993. PA0 8. "Vitamin E consumption and the Risk Coronary heart Disease in Men" Rimm, EE. B. New Engl. J. Med. 328:1450-1456, 1993. PA0 9. "Antioxidant Vitamins and Coronary Heart Disease" The New England Journal of Medicine Val. 323, pp. 1487-1489, 1993. PA0 10. "Antioxidant-Mediated Inhibition of Macrophage Modification of low density Lipoprotein" Life Chemistry Reports, 1994, Vol. 12, pp. 69-78.
None of these publications has examined the effect of egg PUFA on plasma LDL. The publication of Parthasarathy et al., which was performed with a special sunflower oil (Trisun 80) comprising a low amount of PUFA (8% of lineolic acid) shows that due to this fact inhibition of LDL oxidation is achieved. However, as is readily understood, an experiment performed with oil in feeding rabbits cannot be conclusive for the production of a functional egg.
Moreover, in these publications there is no indication given whatsoever that the addition of carotenoids and of other antioxidants would increase resistance of the LDL against oxidation.
As is well known in the nutrition science in each condition and diet each factor has to be considered on its own merits.
As cholesterol is very reactive to oxidation and as peroxidation is a chain reaction, a lower amount of antioxidative materials are required for protecting the cholesterol at the initial phases than later on. It could therefore be expected ingesting "Protected" cholesterol, namely cholesterol in an environment of low oxidative stress, e.g., comprising a large amount of high antioxidants and low PUFA, which may become "Pro-oxidants", could give a significant impact in the plasma LDL oxidizibility.
Thus, an egg enriched with antioxidants, known for the LDL protection potential and low PUFA can provide such "protecting" environment.
Indeed some research papers show that the reactivity of LDL to oxidation is determined not only by its antioxidant content but also by other compositional factors and more specifically by the ratio of oleic acid content to linoleic acid content (See: oxidation resistance, oxidation rate and extent of oxidation of human low density lipoprotein depend on the ratio of oleic acid content to linoleic acid content: studies in vitamin E deficient subjects; Kleinveld et al.; Free radic. med. 1993 SIP 15 (3) 273-80).
Although chicken diet may greatly affect the fatty acid profile of the eggs, the question is how much this can influence the LDL in the consumer. It was shown that when primates on high cholesterol diet received various amounts of FA, that the FA profile in LDL was indeed influenced by the diet but linoleic acid was the predominant PUFA in all of the LDLs. The rates of LDL oxidation were linearly dependent upon the concentration of PUFA.
The final proof that PUFA and mainly linoleic acid are involved in LDL oxidation was recently shown by the analysis of the lipid oxidation products in the oxidized LDL following immunological activation of the human monocytes: the major FA oxidation product was esterified hydroperoxyoctadecadienoic acid (HPODE) which is the oxidized product of the main PUFA in human LDL-linoleic acid.
It has thus been desirable to devise an egg comprising a synergistic composition of antioxidants preventing the oxidation of LDL. The production of such an egg preferably use as many as possible ingredients of standard chicken mixtures. The method should be simple and should not require many changes by the manufacturer of the mixture or by the farmer growing the chickens.
It is well known that various egg components are affected by the chicken feed, e.g., vitamins E, A and other vitamins. See: Modifying Vitamin Composition of eggs: A review by E. C. Naber. J. Applied poultry res. 2:385-393, 1993.
Recently a paper ".alpha.-tocopherol, .beta.-carotene and retinol enrichment of chicken eggs" Jiang, Y. H., et al., Poult SCI, 1994, Jul.:73(7):11137-43, showed that it is possible to enrich these components in the egg significantly. However, supplemental .beta.-Carotene may markedly decrease the yolk deposition of vitamin E. Moreover, .beta.-carotene is most intensively transformed in the chicken to vitamin A and only traces of it attain the yolk compared to the rapid and effective deposition of dietary oxycarotenoids.
The kind and concentration of oxycarotenoids in the yolk is strongly influenced by the diet, i.e., the carotenoid concentration, protective antioxidants, destructive factors, e.g., proxidants such as PUFA, storage and processing conditions, etc. Oxycarotenoids are considered in relation to their pigmentation attributes, since they contribute most of the yolk pigments. .beta.-carotene is thus not the selected antioxidant for chicken feed. Moreover, it is known that oxycarotenoids which are readily deposited in the yolk perform an antioxidative function in the LDL.
In the present invention those principles were applied to obtain an egg comprising more iodine, carotenoids and vitamin E.
A recent research on antioxidants showed that adding vitamin E which increased LDL content 2.5 times the baseline amount, reduced the reactivity of LDL to oxidation by 50%. See: Effect of dietary antioxidant combinations in humans, protection of LDL by vitamin E but not by .beta.-carotene, Reaven et al., 1933, Arterioscler-Thromb Apr. 13(4)590-600.
As in the process of oxidation-protection, vitamin E itself is consumed, and thus its concentration should be increased in proportion to PUFA. The generally accepted ratio being between 0.4-0.6 mg vitamin E/1 gram PUFA.
Moreover, recent research has raised the question regarding the potential of oxidized vitamins (antioxidants) becoming a proxidant and then facilitating oxidation. Thus, it might not be enough to increase the amount of vitamin E but rather provide further protection, e.g. by carotenoids, vitamin C, flavonoids and/or other antioxidants, to create a synergistic effect in antioxidative process.
Vitamin E also protects other antioxidants, e.g., carotenoids. Thus, it enhances pigmentation in the yolk (See: "Oxycarotenoids in Poultry Feeds, Carotenoids as Colorants and Vitamins as Precursor" Marusich and Bauernfeind, Academic Press 1981, pages 319-444). This results in a considerable increase of the xanthophyll concentration in blood plasma (See: Carotens and other Vitamin A Precursors in Animal Feed, Bauernfeind et al., Carontenoids as Colorants and Vitamin A Precursors. Academic Press 1981). Other antioxidants were also effective in the same manner, i.e., ethoxyquin (EMQ) and butylated hydroxytoluene (BHT) are also known to reduce the oxidative destruction of unsaturated molecules such as PUFA and carotenoids, and thus improve pigmentation (see: Carotenoids--their Nature and Significance in Animal Feeds by T. Latsch, Dept. Of Animal Nutrition and Health, HOFFMANN LA ROCHE LTD., BASEL, SWITZERLAND, 1990). The above antioxidants are synthetic compounds and do not have any nutritional value, but they can contribute to reduce the LDL oxidation.
As indicated above, the LDL is usually rich with PUFA which is mainly C-81:2 (linoleic acid) which is very reactive in the oxidation of LDL. On the other hand the presence of a large amount of MUFA, e.g., C-18:1, oleic acid contributes to the increase of the LDL stability as far as oxidation of LDL is concerned. Also SFA are rather resistant to oxidation.
As cam be understood from the above, the PUFA are very sensitive to oxidation and therefore increase the risk to the LDL oxidation. Thus, one major aspect of the present invention is to reduce the amount of the PUFA present in the egg.